High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Abstract

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy.

NK cell-based immunotherapy for malignant diseases

“…HSCs can be harvested from UCB for clinical applications by using the CliniMACS system to select CD34 1 cells. [153][154][155] Enriching CD34 1 cells from thawed UCB was optimized by using the EloHAES separation method within the CliniMACS system. The CD34 1 cell-derived NK cells were generated in static cell culture bags or in an automated bioreactor in order to produce clinical-grade NK cells in a closed environment.…”

Ex Vivo Generated Natural Killer Cells Acquire Typical Natural Killer Receptors and Display a Cytotoxic Gene Expression Profile Similar to Peripheral Blood Natural Killer Cells

“…Obtaining adequate purity and cell numbers of functional NK cells remains the biggest challenge for their therapeutic use. Recently, we have described a cytokine-based culture method with the capability of generating clinically relevant NK cell products from umbilical cord blood (UCB)-derived hematopoietic stem cells [9,10], obtaining high cell numbers, purity, and functionality. Currently, NK cell products generated by this method are investigated in a phase I clinical trial to treat elderly acute myeloid leukemia patients in a nontransplantation setting (Dutch Trial Register NTR2818) [11,12].…”

“…CD34 + UCB cells were transferred into culture plates and expanded and differentiated according to culture method III as described previously [9]. In short, CD34…”

“…Initially, we further characterized the cellular composition of our recently developed cytokine-based expansion and differentiation method [9] for generating NK cells from CD34 + hematopoietic stem cells (Fig. 1A).…”

Cellular engineering and therapy in combination with cord blood allografting in pediatric recipients

“…To overcome these limitations, many groups have developed techniques for ex vivo expansion of NK cells adapted to UCB as the starting source to enable adoptive immunotherapy. [39][40][41][42][43][44][45][46][47][48] In addition, ex vivo IL-2-expanded NK cells from CB were shown to be active against AML blasts and showed anti-leukemia activity in vivo when infused into mice bearing human AML. 49 NK cell-based cancer immunotherapy is an expanding scientific area of investigation.…”